PROJECT SUMMARY Arrhythmia-induced sudden cardiac death claims more than 250,000 lives each year in the United States. A subset of these deaths result from highly penetrant inherited arrhythmia syndromes, such as Brugada Syndrome (BrS). Loss of function variants in the voltage-gated cardiac sodium channel, SCN5A, are the major known genetic cause of BrS. Additionally, regulatory variation that affects SCN5A expression has been implicated in BrS. If BrS is diagnosed, sudden cardiac death can often be averted with an implantable cardioverter-defibrillator. Therefore the American College of Medical Genetics recommends that incidental pathogenic variants in SCN5A be reported so that patients and family members can be accurately diagnosed and treated. Unfortunately, we and others have found that the pathogenicity of SCN5A variants is often unknown or disputed and often does not accurately predict arrhythmias. Improved classification of coding and non-coding SCN5A variants as pathogenic or benign would enable more accurate diagnosis and treatment of BrS. My hypothesis is that in vitro high-throughput screening methods can accurately identify a broad set of pathogenic coding and regulatory SCN5A loss of function variants. I will pursue two specific aims to test this hypothesis: 1) Identify SCN5A coding variants that reduce channel activity and trafficking, and 2) Identify enhancers and functional non-coding SNPs affecting SCN5A expression. Under the first aim, I will survey the activity and trafficking of the 1920 possible coding variants in an important 96 amino acid region of SCN5A. My preliminary data shows proof of principle experiments that demonstrate the feasibility of mutagenesis, transgenesis, and functional assay methods necessary to complete this screen. Under the second aim, I have implemented a high-throughput sequencing-based screen to discover enhancers that affect SCN5A expression. I propose to finish this enhancer screen, then test whether SNPs that affect these enhancers' activity contribute to BrS. These studies are innovative because they leverage recently developed high-throughput sequencing-based methods to broaden and improve our understanding of variants in an important disease gene. As genomic medicine continues to become more commonplace, the challenge of interpreting patients' variants will continue to grow. This project provides a template for a general approach for improving the breadth and quality of genomic annotations to help deliver on the promise of genomic and precision medicine.